title: Distribution of epithelial ankyrin (Ank3) spliceoforms in renal proximal and distal tubules.
abstract: In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3(200/215). To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3(200/215) but no Ank3(170) or Ank3(120). Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3(200/215). These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3(200/215) and Na-K-ATPase abundance measured a lower Ank3(200/215)-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1 cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3(200/215) suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.
authors.affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: R B
authors.initials: RB
authors.lastname: Doctor
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: L L
authors.initials: LL
authors.lastname: Peters
authors.forename: S E
authors.initials: SE
authors.lastname: Lux
authors.forename: L J
authors.initials: LJ
authors.lastname: Mandel
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D017487:Ankyrins / Q000032:analysis / Q000096:biosynthesis*; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007672:Kidney Cortex / Q000166:cytology / Q000378:metabolism; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D051381:Rats
chemical_list: C094681:Ank3 protein, mouse; C490614:Ank3 protein, rat; D017487:Ankyrins